Dark-field microscope apparatus utilizing portable electronic communication device

ABSTRACT

A mobile phone-based dark field microscope (MDFM) apparatus suitable for quantifying nanoparticle signals is provided. The MDFM apparatus includes an electrically operated light source, a dark-field condenser, a slide housing configured to receive an analytical slide, and an adapter housing configured to receive an objective lens and receive a portable electronic communication device. The slide housing positions the analytical slide between the objective lens and the dark-field condenser. The adapter housing registers the objective lens with a camera lens of the portable electronic communication device. A method for performing a biological quantitative study using the dark-field microscope apparatus is further provided.

STATEMENT OF RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national phase filing ofInternational Application No. PCT/US2018/046003 filed Aug. 9, 2018, andclaims priority to U.S. Provisional Patent Application No. 62/542,980filed Aug. 9, 2017, wherein the entire contents of the foregoingapplications are hereby incorporated by reference herein.

GOVERNMENT RIGHTS IN INVENTION

This invention was made with government support under R01 AI113725 andR01 AI122932 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The present disclosure relates to dark-field microscope apparatuses andmethods of performing biological quantitative studies utilizing suchapparatuses, including (but not limited to) dark-field microscopeapparatuses and methods suitable for quantifying nanoparticle signalsthat may be generated with nanoparticles providing scatter signals(e.g., gold or silver nanoparticles such as nanorods).

BACKGROUND

Gold nanoparticles have become prevalent labeling agents for detectionand quantification of different targets, cellular imaging, biomolecularquantification, and performance of interaction studies. Conventionalstudies utilizing gold nanoparticles rely on complex equipment thatlimits their applicability to field settings. Portable spectrometry hasbeen nominated as a potential solution for nanoparticle quantification;however, it suffers from complex setup requirements as well as eitherlow throughput or low sensitivity. Nanoparticle-based lateral-flowchromatographic immunoassays are point-of-care devices, but usually arenot quantitative, and require extensive development and validation.

Nanoparticle-based variants of standard immunoassays have potential asquantitative point-of-care immunoassays, since antibody-linkednanoparticle probes can be stored dry at ambient temperature (unlikeenzyme-linked antibodies used in conventional immunoassays, whichrequire low temperature storage).

Dark-field microscopy (also known as “dark-ground microscopy”) describesmicroscopy methods that exclude the unscattered beam from the image. Asa result, the field around the specimen (i.e., where there is nospecimen to scatter the beam) is generally dark. Dark-field microscope(DFM) image analysis is commonly used to sensitively detect andprecisely quantify nanoparticle-based immunoassay variants. The pairingof DFM image analysis and nanoparticles has resolved many criticalquantification problems in bioresearch and clinical practice. DFM imageanalysis requires a high-magnification microscope, sincelow-magnification (far-field) DFM images are highly sensitive to surfaceartifacts and debris that can easily mask nanoparticle signals. Thesize, cost, and delicate character of conventional DFM systems limittheir utility in non-laboratory settings—such as field hospitals andother settings, in which these factors represent barriers to their use.In addition to their lack of portability, conventional DFM systems mayalso be limited in terms of their ease of use.

Attempts to develop more portable DFM approaches date back to 1958, whendermatologists utilized DFM image analysis to diagnose agentsresponsible for multiple diseases (including syphilis) that producedskin lesions, but these devices fell out of use upon the development ofother technologies, and few advances in DFM image analysis have beenmade since that time.

Recent technology advances driven largely by mobile phone cameradevelopment have spurred the use of mobile phone cameras in medicalapplications, including portable microscopy for numerous point-of-carediagnostics. As of the effective date of this application, however,Applicant is unaware of any far-field DFM system incorporating a mobilephone camera.

SUMMARY

Disclosed herein is a mobile phone-based DFM (MDFM) apparatus suitablefor quantifying nanoparticle signals for a variety of research andmedical applications. Such apparatus is lightweight and portable incharacter. In certain embodiments, a MDFM apparatus uses an inexpensivetriple-LED light source, a standard dark-field condenser, an objectivelens (e.g., 20× magnification, 10× magnification, or any other suitablemagnification), and structural elements (e.g., one or more housings)configured to mate these components to a mobile phone camera. MDFMapparatuses disclosed herein are compatible with high throughput assays,and provide robust sensitivity, stability, and reproducible results withsimple setup. Such apparatuses may provide a valuable platform for thepractice of nanotechnology in field settings and other resource-limitedenvironments.

In one aspect, the present disclosure relates to a dark-field microscopeapparatus including: an adapter housing, an electrically operated lightsource, a dark-field condenser, and a slide housing. The adapter housingis configured to receive a portable electronic communication device andan objective lens, and to cause the objective lens to be registered witha camera lens of the portable electronic communication device when theportable electronic communication device is received by the adapterhousing. The dark-field condenser is configured to condense lightemissions generated by the electrically operated light source. The slidehousing is configured to receive at least a portion of an analyticalslide and position the analytical slide between the dark-field condenserand the objective lens.

In another aspect, a biomolecule quantification device comprises thedark-field microscope apparatus as disclosed herein, wherein theanalytical slide is received by the slide housing, and the analyticalslide contains at least one nanoparticle-based biomarker. In certainembodiments, the at least one nanoparticle-based biomarker comprises atleast one gold or silver nanoparticle.

In another aspect, a method for performing a biological quantitativestudy utilizes a dark-field microscope apparatus as disclosed herein.The method includes: inserting at least a portion of the analyticalslide into the slide housing to position the analytical slide betweenthe dark-field condenser and the objective lens, wherein the analyticalslide comprises at least one biomolecule and at least onenanoparticle-based biomarker; transmitting light emissions generated bythe electrically operated light source through the dark-field condenserto impinge condensed light emissions on a target region of the at leasta portion of the analytical slide; and generating a magnified image ofthe target region using the objective lens and the portable electroniccommunication device received by the adapter housing.

In another aspect, the present disclosure relates to a dark-fieldmicroscope apparatus including: an objective lens; a light source; adark-field condenser configured to condense light emissions generated bythe light source; a slide housing configured to receive an analyticalslide and position the analytical slide between the dark-field condenserand the objective lens; and an adapter housing configured to receive aportable electronic communication device and to receive the objectivelens, and configured to register the objective lens with a camera lensof the portable electronic communication device.

In another aspect, the present disclosure relates to a nanoparticlequantification device comprising a dark-field microscope apparatus asdisclosed herein, wherein the analytical slide is received by the slidehousing, and at least one type of nanoparticle is supported on or abovea surface of the analytical slide.

In another aspect, the present disclosure relates to a biomoleculequantification device comprising a dark-field microscope apparatus asdisclosed herein, wherein the analytical slide is received by the slidehousing, and at least one nanoparticle-conjugated biomarker and acorresponding binding target are supported on or above a surface of theanalytical slide.

In another aspect, the present disclosure relates to a method forperforming a biological quantitative study utilizing a dark-fieldmicroscope apparatus as disclosed herein, the method comprising:inserting at least a portion of the analytical slide into the slidehousing to expose an area of interest of the analytical slide to anoptical path between the dark-field condenser and the objective lens,wherein the area of interest of the analytical slide contains targetbiomolecules and labels embodying conjugated nanoparticles comprisingbinding counterparts for the target biomolecules; transmitting lightemissions generated by the light source through the dark-field condenserto impinge condensed light on the area of interest on the analyticalslide; and generating a magnified image of the area of interest on theanalytical slide using the objective lens and the portable electroniccommunication device received by the adapter housing.

In another aspect, the present disclosure relates to a method fordiagnosing a disease, the method comprising: inserting at least aportion of an analytical slide into the slide housing of a dark-fieldmicroscope apparatus as disclosed herein to expose an area of interestof the analytical slide to an optical path between the dark-fieldcondenser and the objective lens, wherein the area of interest of theanalytical slide contains target biomolecules and labels embodyingconjugated nanoparticles comprising binding counterparts for the targetbiomolecules; transmitting light emissions generated by the light sourcethrough the dark-field condenser to impinge condensed light on the areaof interest on the analytical slide; generating a magnified image of thearea of interest on the analytical slide using the objective lens andthe portable electronic communication device received by the adapterhousing; and analyzing the magnified image.

In another aspect, any one or more aspects or features described hereinmay be combined with any one or more other aspects or features foradditional advantage.

Other aspects and embodiments will be apparent from the detaileddescription and accompanying drawings.

Those skilled in the art will appreciate the scope of the presentdisclosure and realize additional aspects thereof after reading thefollowing detailed description of the preferred embodiments inassociation with the accompanying drawing figures.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view of light interacting components of adark-field microscope apparatus without illustration of an adapterhousing or slide housing according to the present disclosure, showingpassage of a light beam between the components.

FIG. 2A is an exploded, lower perspective view of components of a mobilephone-based dark-field microscope (MDFM) apparatus according to oneembodiment of the present disclosure.

FIG. 2B is an upper perspective view of an assembled MDFM apparatusincluding the components shown in FIG. 2A, with a mobile phone receivedby the adapter housing.

FIG. 3 provides a comparison of images obtained at high and lowmagnification for biological moieties with and without nanoparticles,respectively, obtained via a MDFM microscope system, a desktopdark-field microscope system (DDFM), and a bright field microscopysystem, with 20× and 10× objective lenses employed for MDFM, and with10× and 4× objective lenses employed for DDFM and bright fieldmicroscopy.

FIG. 4 shows six MDFM images captured with a 10× objective lens atdifferent working distances of 1 mm, 3 mm, 5 mm, 7 mm, 9 mm, and 11 mm.

FIG. 5 is a plot of MDFM magnification (M) with a 10× objective lensversus working distance (d, mm), with a superimposed linear correlationplot and coefficient of determination (R²) value.

FIG. 6 shows two MDFM images captured with a 20× objective lens at 2.5mm and 3.5 mm working distances,

FIG. 7A is a schematic view of a binding affinity assay scheme involvingbinding between a carboxyl-acid functionalized gold nanorod (AuNR) andan amine modified slide.

FIG. 7B provides superimposed plots of response versus nanoparticleconcentration (μg/μL) for a binding affinity assay according to thescheme of FIG. 7A obtained using a MDFM apparatus as disclosed hereinand a conventional DDFM system, with corresponding nonlinear fittedcurves and K_(d) values, wherein data points represent the mean±SEM of 5sample replicates.

FIG. 8A is a schematic view of a protein quantification assay schemeinvolving quantification of a biotinylated antibody aCD9 tagged with anavidin modified gold nanoparticle (AuNR) and bound to a protein A/Gmodified slide.

FIG. 8B provides superimposed plots of response versus targetconcentration (μg/μL) for a protein quantification assay according tothe scheme of FIG. 8A obtained using a MDFM apparatus as disclosedherein and a DDFM system, with corresponding linear fits and coefficientof determination (R²) values, and with data points representing themean±SEM of 5 sample replicates.

FIG. 9 is a table identifying sensitivity (including limit of detection(LOD) and limit of quantitation (LOQ) values) for the binding affinityand protein quantification assays described in connection with FIGS. 7Ato 8B.

FIG. 10 is a table comparing features of a MDFM system as disclosedherein and a DDFM system.

DETAILED DESCRIPTION

The embodiments set forth below represent the necessary information toenable those skilled in the art to practice the embodiments andillustrate the best mode of practicing the embodiments. Upon reading thefollowing description in light of the accompanying drawing figures,those skilled in the art will understand the concepts of the disclosureand will recognize applications of these concepts not particularlyaddressed herein. It should be understood that these concepts andapplications fall within the scope of the disclosure and theaccompanying claims.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, these elements should notbe limited by these terms. These terms are only used to distinguish oneelement from another. For example, a first element could be termed asecond element, and, similarly, a second element could be termed a firstelement, without departing from the scope of the present disclosure. Asused herein, the term “and/or” includes any and all combinations of oneor more of the associated listed items.

It will be understood that when an element is referred to as being “on”or extending “onto” another element, it can be directly on or extenddirectly onto the other element or intervening elements may also bepresent. In contrast, when an element is referred to as being “directlyon” or extending “directly onto” another element, there are nointervening elements present. Likewise, it will be understood that whenan element is referred to as being “over” or extending “over” anotherelement, it can be directly over or extend directly over the otherelement or intervening elements may also be present. In contrast, whenan element is referred to as being “directly over” or extending“directly over” another element, there are no intervening elementspresent. It will also be understood that when an element is referred toas being “connected” or “coupled” to another element, it can be directlyconnected or coupled to the other element or intervening elements may bepresent. In contrast, when an element is referred to as being “directlyconnected” or “directly coupled” to another element, there are nointervening elements present.

Relative terms such as “below” or “above” or “upper” or “lower” or“horizontal” or “vertical” may be used herein to describe a relationshipof one element or region to another element or region as illustrated inthe Figures. It will be understood that these terms and those discussedabove are intended to encompass different orientations of the device inaddition to the orientation depicted in the Figures.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the disclosure.As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises,”“comprising,” “includes,” and/or “including” when used herein specifythe presence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this disclosure belongs. It willbe further understood that terms used herein should be interpreted ashaving a meaning that is consistent with their meaning in the context ofthis specification and the relevant art and will not be interpreted inan idealized or overly formal sense unless expressly so defined herein.

Disclosed herein is a mobile phone-based DFM (MDFM) apparatus suitablefor quantifying nanoparticle signals for a variety of research andmedical applications. Such apparatus is lightweight and portable incharacter. In certain embodiments, a MDFM apparatus uses an inexpensivetriple-LED light source, a standard dark-field condenser, an objectivelens (e.g., 20× magnification, 10× magnification, or any other suitablemagnification), and structural elements (e.g., one or more housings)configured to mate these components to a mobile phone camera. MDFMapparatuses disclosed herein are compatible with high throughput assays,and provide robust sensitivity and stability with simple setup, therebyproviding a valuable platform for the practice of nanotechnology infield settings and other resource-limited environments.

Binding affinity and protein targeting studies conducted in parallelamong MDFM apparatuses and desktop DFM systems validated thequantification capability of the proposed mobile MDFM platform. Incertain embodiments, a MDFM apparatus may weigh less than about 400 g(e.g., about ˜380 g) and cost less than $2000 including the mobilephone, while achieving performance analogous to that of a standarddesktop dark-field microscope for quantifying target biomolecules invarious assay schemes. MDFM apparatuses as disclosed herein allowstable, nanoparticle-based quantitation assays to be performed inresource-limited areas where standard assay approaches were previouslyimpractical. In at least certain embodiments, MDFM apparatuses disclosedherein exhibit higher linearity, similar sensitivity, and similarstability in comparison to desktop DFM (DDFM) systems, and may be usedin the performance of various bioassays, including high throughputassays and/or assays utilizing nanoparticle labeling. Analysis of imagescaptured with MDFM apparatuses as disclosed herein reveal similarnanoparticle quantitation results to images acquired with a much largerand more expensive desktop DFM system.

Certain embodiments are directed to a dark-field microscope apparatusincluding: an adapter housing, an electrically operated light source, adark-field condenser, and a slide housing. The adapter housing isconfigured to receive a portable electronic communication device and anobjective lens, and to cause the objective lens to be registered with acamera lens of the portable electronic communication device when theportable electronic communication device is received by the adapterhousing. The dark-field condenser is configured to condense lightemissions generated by the electrically operated light source. The slidehousing is configured to receive at least a portion of an analyticalslide and position the analytical slide between the dark-field condenserand the objective lens. In certain embodiments, the adapter housing andthe slide housing may be fabricated via three-dimensional printing,molding, machining, or other additive material addition and/orsubtractive material removal processes. In certain embodiments,materials for fabricating the adapter housing and the slide housing mayinclude one or more polymeric, metal, composite, and/or other materials.In certain embodiments, the adapter housing and the slide housing maycomprise black acrylonitrile-butadiene-styrene (ABS) material.Preferably, the adapter housing and the slide housing are fabricated ofmaterials sufficiently dimensioned to block transmission of ambientlight.

Although the terms “MDFM” and “mobile phone dark-field microscope” areused in the present disclosure, it is to be appreciated that suchapparatuses are not limited to the use of mobile phones, and may utilizevarious types of portable electronic communication devices thatincorporate cameras—whether or not such devices necessarily embodymobile phones. For example, various tablet or tablet-like devices (e.g.,Apple IPOD®, Apple IPAD®, and the like) that incorporate sophisticatedcameras and processing capability, and are capable of WiFicommunications without necessarily including cellular phone capability,may be used. Additionally, numerous types and brands of mobile phonesincorporating cameras may be used, including various models produced bymanufacturers such as (but not limited to) Apple, Samsung, Google,Huawei, ZTE, Lenovo, LG, Motorola, Sony, Nokia, and the like.

In one aspect, the present disclosure relates to a dark-field microscopeapparatus including: an adapter housing, an electrically operated lightsource, a dark-field condenser, and a slide housing. The adapter housingis configured to receive a portable electronic communication device andan objective lens, and to cause the objective lens to be registered witha camera lens of the portable electronic communication device when theportable electronic communication device is received by the adapterhousing. The dark-field condenser is configured to condense lightemissions generated by the electrically operated light source. The slidehousing is configured to receive at least a portion of an analyticalslide and position the analytical slide between the dark-field condenserand the objective lens.

In certain embodiments, the adapter housing comprises a main body and alens receiver that protrudes from the main body; the lens receiver isconfigured to receive the objective lens; and the slide housing isconfigured to receive at least portions of the lens receiver and theobjective lens.

In certain embodiments, the main body comprises a support surfaceconfigured to abut a face of the portable electronic communicationdevice, and the main body comprises at least one lateral wall configuredto abut at least one lateral edge of the portable electroniccommunication device.

In certain embodiments, the slide housing comprises a bore; the lensreceiver comprises a first outer wall configured to fit into a firstportion of the bore; and the dark-field condenser comprises a secondouter wall configured to fit into a second portion of the bore. Incertain embodiments, the bore, the first outer wall, and the secondouter wall may be generally tubular in shape. Such tubular shape mayhave a round, elliptical, square, or other suitable cross-sectionconformation in certain embodiments.

In certain embodiments, the dark-field microscope apparatus furtherincludes: a first set screw configured to selectively promote engagementbetween the lens receiver and either (i) the slide housing or (ii) theobjective lens, to adjust a first distance between the objective lensand the analytical slide; and a second set screw configured toselectively promote engagement between the slide housing and thedark-field condenser, to adjust a second distance between the dark-fieldcondenser and the analytical slide.

In certain embodiments, at least one of the slide housing or the adapterhousing is configured to permit a working distance between the objectivelens and the analytical slide to be adjusted, and the dark-fieldmicroscope apparatus further comprises at least one locking element thatis selectively operable to fix the working distance between theobjective lens and the analytical slide.

In certain embodiments, the dark-field microscope apparatus furtherincludes the objective lens. In certain embodiments, the objective lens,the slide housing, and the dark-field condenser are configured to forman optical path having a center aligned with an emissive center of thelight source.

In certain embodiments, the slide housing defines at least one slot thatis configured to receive at least a portion of the analytical slide. Theslide housing is configured to permit the analytical slide to moverelative to the slide housing to expose a different portion of theanalytical slide to the optical path with each movement of theanalytical slide.

In certain embodiments, the dark-field microscope apparatus furtherincludes a base element configured to support the electrically operatedlight source and configured to receive an end portion of the slidehousing.

In certain embodiments, the objective lens provides a magnification ofat least 10 times. In certain embodiments, the objective lens isconfigured to provide variable magnification (i.e., multiple differentmagnifications).

In certain embodiments, the electrically operated light source comprisesa solid state light source, such as a battery-powered solid state lightsource.

In certain embodiments, the electrically operated light source comprisesat least one light emitting diode. In certain embodiments, the at leastone light emitting diode is configured to generate a peak wavelength inthe visible range.

In certain embodiments, the portable electronic communication devicecomprises a mobile phone.

In certain embodiments, the adapter housing and the slide housing eachcomprise at least one polymeric material.

In certain embodiments, the adapter housing and the slide housing arefabricated by three-dimensional printing.

In certain embodiments, the adapter housing and the slide housing arefabricated by molding.

In certain embodiments, the adapter housing and the slide housing arefabricated by a subtractive material removal process.

In another aspect, a biomolecule quantification device comprises thedark-field microscope apparatus as disclosed herein, wherein theanalytical slide is received by the slide housing, and the analyticalslide contains at least one nanoparticle-based biomarker. In certainembodiments, the at least one nanoparticle-based biomarker comprises atleast one gold or silver nanoparticle.

In another aspect, a method for performing a biological quantitativestudy utilizes a dark-field microscope apparatus as disclosed herein.The method includes: inserting at least a portion of the analyticalslide into the slide housing to position the analytical slide betweenthe dark-field condenser and the objective lens, wherein the analyticalslide comprises at least one biomolecule and at least onenanoparticle-based biomarker; transmitting light emissions generated bythe electrically operated light source through the dark-field condenserto impinge condensed light emissions on a target region of the at leasta portion of the analytical slide; and generating a magnified image ofthe target region using the objective lens and the portable electroniccommunication device received by the adapter housing.

In certain embodiments, the foregoing method may utilize one or moregold or gold-containing nanoparticles.

In another aspect, the present disclosure relates to a dark-fieldmicroscope apparatus including: an objective lens; a light source; adark-field condenser configured to condense light emissions generated bythe light source; a slide housing configured to receive an analyticalslide and position the analytical slide between the dark-field condenserand the objective lens; and an adapter housing configured to receive aportable electronic communication device and to receive the objectivelens, and configured to register the objective lens with a camera lensof the portable electronic communication device.

In certain embodiments, the objective lens, the slide housing, and thedark-field condenser are configured to form an optical path having acenter aligned with an emissive center of the light source.

In certain embodiments, the slide housing defines at least one slot toreceive the analytical slide; and the slide housing is configured topermit the analytical slide to move relative to the slide housing toexpose a different portion of the analytical slide to the optical pathwith each movement of the analytical slide.

In certain embodiments, the dark-field microscope apparatus isconfigured to permit the objective lens to be swapped with a differentobjective lens to provide multiple different magnifications.

In certain embodiments, the objective lens is configured to providevariable magnification.

In certain embodiments, the objective lens comprises at least one lensproviding a magnification value in a range of from 4 times to 100 times.

In certain embodiments, at least one of the slide housing or the adapterhousing is configured to permit a working distance between the objectivelens and the analytical slide to be adjusted. In certain embodiments,the dark-field microscope apparatus further includes at least onelocking element that is selectively operable to fix the working distancebetween the objective lens and the analytical slide.

Exemplary MDFM Apparatus

FIG. 1 is a perspective schematic view of components of a MDFM apparatus10 without illustration of an adapter housing or slide housing accordingto the present disclosure, according to one embodiment of the presentdisclosure. Various functional parts of the MDFM apparatus include amobile phone 12 or other portable electronic communication device (whichincludes an integrated camera lens 14 and an imaging sensor 16), anobjective lens 18, a dark-field condenser 22, and a light source 24,wherein a slide 20 including one or more sample-containing regions 25may be positioned between the objective lens 18 and the dark-fieldcondenser 22. In certain embodiments, the imaging sensor 16 may includea charge-coupled device (CCD) or a CMOS sensor. In certain embodiments,the light source 24 includes an emissive center 23 and may include oneor more solid state emitters (e.g., light emitting diodes), and may bebattery powered to facilitate portability of the MDFM apparatus 10.Emissions from the light source 24 form light a beam 26A that istransmitted through the dark-field condenser 22 to form a condensedlight beam 26B that illuminates one or more portions of the slide 20containing one or more samples in the sample-containing regions 25.Portions of the condensed light beam 26B that transit through the slide20 and the sample(s) form an exit beam 26C that is focused by theobjective lens 18 to form a focused exit beam 26D and received by themobile phone 12 or other portable electronic communication device, withthe focused exit beam 26D being transmitted through the integratedcamera lens of 14 the mobile phone 12 to impinge on the imaging sensor16. The integrated camera lens 14 of the mobile phone 12 may be fixed orvariable in character. Although FIG. 1 does not illustrate an adapterhousing or slide housing, and FIG. 1 shows various components as beingspaced apart from one another (to permit illustration of passage oflight through the components), it is to be appreciated than an operativeMDFM apparatus may include an adapter housing and a slide housing, andcomponents of the MDFM apparatus may be positioned much closer (or incontact with) one another, such as (but not limited to) a first distanceD1 between the objective lens 18 and the analytical slide 20, and asecond distance D2 between the dark-field condenser 22 and theanalytical slide 20.

FIG. 2A is an exploded perspective view illustration of components of aMDFM apparatus 30 according to one embodiment of the present disclosure.The MDFM apparatus 30 includes a base element 32, an electricallyoperated light source 36, a dark-field condenser 38, a slide housing 44(having a generally cylindrical shape), an adapter housing 70, and anobjective lens 62 received within a portion of the adapter housing 70.Starting at the bottom of the figure, FIG. 2A shows a generallycylindrical base element 32 configured to receive the electricallyoperated light source 36, which may include one or more LEDs optionallyconfigured to generate a peak wavelength in the visible range. The baseelement 32 defines a vertical slot 34 that opens to a cylindrical cavity(not shown), and includes a lateral retention element 33 that may beused to promote retention of the slide housing 44 within a portion ofthe cavity of the base element 32. In one embodiment, the lateralretention element 33 includes a tapped (i.e., threaded) opening that maybe configured to receive a threaded screw (not shown) that may bethreaded through the tapped opening to selectively engage a cylindricallower portion 48 of the slide housing 44 when the slide housing 44 isreceived by the base element 32. The dark-field condenser 38 includes agenerally cylindrical body with a radially protruding lip 40 along alower edge thereof. The dark-field condenser 38 also defines a loweraperture 42 configured to receive emissions of the electrically operatedlight source 36 when the dark-field condenser 38 is positioned above thelight source 36. The dark-field condenser 38 is also configured to bereceived within a lower aperture 54 defined by the cylindrical lowerportion 48 of the slide housing 44. The slide housing 44 defines atleast one (e.g. horizontally arranged) slot 52 configured to receive ananalytical slide 60. The slide housing 44 includes a cylindrical upperportion 46 and a cylindrical lower portion 48 with a shoulder 50therebetween, with the lower portion 48 having a larger outer diameterthan the upper portion 46. The upper portion 46 of the slide housing 44defines an upper aperture (not shown) configured to receive at least aportion of the objective lens 62, which may also be received by adownwardly protruding, lower portion of an adapter housing 70 thatembodies a lens receiver 64. The upper portion 46 of the slide housing44 also includes a radially extending protrusion 58 that may include atapped opening (not shown) for receiving a set screw (as shown in FIG.2B). The lens receiver 64 of the adapter housing 70 includes a proximalportion 66 and a distal portion 68 that define a cavity which receivesthe objective lens 62. In certain embodiments, the proximal portion 66has a larger diameter than the distal portion 68 of the lens receiver64. In certain embodiments, the objective lens 62 provides at least 10×magnification (e.g., 10×, 15×, 20×, 25×, 30×, or some other desiredmagnification value, wherein in certain embodiments 4× or 8×magnification may be used). In certain embodiments, the objective lens62 may include multiple lenses arranged in series. The adapter housing70 is configured to receive a portable electronic communication device(e.g., a mobile phone) as well as the objective lens 62, and to causethe objective lens 62 to be registered with a camera lens of theportable electronic communication device when the portable electroniccommunication device is received by the adapter housing 70. The adapterhousing 70 includes a main body 72 and the lens receiver 64 thatprotrudes downward from the main body 72. The main body 72 of theadapter housing 70 includes a support surface 73 configured to abut aface of the portable electronic communication device, and includes atleast one lateral wall 74 configured to abut at least one lateral edgeof the portable electronic communication device.

With continued reference to FIG. 2A, a lower portion of the slidehousing 44 includes a radially extending protrusion 56 that isconfigured to be received by the vertical slot 34 defined in the baseelement 32. The radially extending protrusion 56 may further include atapped (i.e., threaded) opening configured to receive a set screw (suchas shown in FIG. 2B) configured to set a relative position between theslide housing 44 and the dark-field condenser 38, whereby manualmovement between the slide housing 44 and the dark-field condenser 38,and selective engagement therebetween using a set screw, permitsadjustment of a distance D2′ between the dark-field condenser 38 and theanalytical slide 60. The cylindrical upper portion 46 of the slidehousing 44 includes a sidewall defining a tapped (i.e., threaded)opening configured to receive another set screw (shown in FIG. 2B)configured to selectively promote engagement between the lens receiver64 and the slide housing 44, whereby manual movement between the lensreceiver 64 and the slide housing 44, and selective engagementtherebetween using a set screw, permits adjustment of a distance D1′between the objective lens 62 and the analytical slide 60 received bythe slot 52 defined in the slide housing 44.

FIG. 2B is a perspective view photograph of an assembled MDFM apparatus30 incorporating the components illustrated in FIG. 2A, with a mobilephone 85 received by the adapter housing 70. As shown, the mobile phone85 is arranged between lateral walls 74 and below an upper peripherallip 78 of the adapter housing 70, which includes an open end 79 devoidof a sidewall to permit the mobile phone 85 to be laterally insertedinto the adapter housing 70. FIG. 2B illustrates the slide housing 44supported from below by the electrically operated light source 36arranged within the base element 32, with a lower set screw 80 extendingthrough a tapped opening defined in the radially extending protrusion 56(which extends into the vertical slot 34 of the base element 32) of thecylindrical lower portion 48 to engage the dark-field condenser (notshown) within the lower cylindrical portion 48 of the slide housing 44.An analytical slide 60 is arranged within the slot 52 defined in theslide housing 44, just above the shoulder 50, which represents atransition between the cylindrical lower and upper portions 48, 46 ofthe slide housing 44. As shown, the lens receiver 64 is received withinan aperture 45 defined in the slide housing. FIG. 2B also illustrates anupper set screw 82 extending through a tapped opening in a radiallyextending protrusion 58 (positioned along the cylindrical upper portionof the slide housing 44) to engage the proximal portion 66 of the lensreceiver 64, to enable adjustment of relative position (and the distanceD1′) between the objective lens 62 and the analytical slide 60.

In certain embodiments, the adapter housing and/or the slide housing mayeach comprise at least one polymeric material. In certain embodiments,an opaque polymeric material such as ABS may be used. In certainembodiments, the adapter housing and/or the slide housing may befabricated of metal and/or composite materials. In certain embodiments,various manufacturing techniques such as three-dimensional printing (oranother additive manufacturing process), molding (e.g., injectionmolding), and/or subtractive material removal (e.g., machining) may beused.

In certain embodiments, a biomolecule quantification device comprisesthe dark-field microscope apparatus as disclosed herein, wherein theanalytical slide is received by the slide housing, and the analyticalslide contains at least one nanoparticle-based biomarker. In certainembodiments, the at least one nanoparticle-based biomarker comprises atleast one gold or silver nanoparticle.

In certain embodiments, a method for performing a biologicalquantitative study utilizes a dark-field microscope apparatus asdisclosed herein. The method includes: inserting at least a portion ofthe analytical slide into the slide housing to position the analyticalslide between the dark-field condenser and the objective lens, whereinthe analytical slide comprises at least one biomolecule and at least onenanoparticle-based biomarker; transmitting light emissions generated bythe electrically operated light source through the dark-field condenserto impinge condensed light emissions on a target region of the at leasta portion of the analytical slide; and generating a magnified image ofthe target region using the objective lens and the portable electroniccommunication device received by the adapter housing.

To fabricate an exemplary MDFM apparatus, SolidWorks® 2013 CAD software(Dassault Systemes SolidWorks Corporation) was used to design theadapter housing and slide housing shown in FIGS. 2A and 2B. Suchhousings were fabricated with black acrylonitrile-butadiene-styrene(ABS) using a 3D printing service (3D hubs).

Image Capture and Processing

To provide a basis for comparison, DDFM images were acquired underconsistent lighting and magnification using an Olympus IX81 microscopeequipped with a dark-field condenser, a 4× or 10× magnificationobjective lens, and an Olympus DP71 digital camera, using a 1/45sexposure time. MDFM images using the apparatus described in connectionwith FIGS. 2A and 2B were acquired using a Motorola Moto G2 camera tocapture images from a slide holder case containing a dark-fieldcondenser and a 10× or 20× magnification objective lens and illuminatedwith a constant triple-LED white light source (Modgy, Inc.).Characteristics and components of the DDFM system and MDFM apparatus aredisclosed and compared in tabular form in FIG. 10 .

All images were processed and quantified using a “DarkScatterMaster” DFMalgorithm using the following software input parameters: contourthreshold (Ct)=253.020, center scale (S)=0.8, type=Red, Low (Lt)/High(Ht) quantification limit: 0/62. Motorola Moto G2 (XT1068) images of theMDFM apparatus were captured with a 1/15 s exposure time with OpenCamera (Version 1.32.1) using an ISO 5000 configuration and allowingautofocus and 4× digital zoom. Magnification (M) was defined as thesample image height (h_(i)) divided by the height of the sample object(h_(o)), where h_(o) was the target well diameter (1.5 mm) and h_(i) wasthe diameter of the image in pixels multiplied by the resolution of thesensor chip (72 vs. 432 pixels/inch for MDFM and DDFM, respectively).

Binding Affinity Assay

Carboxyl-functionalized gold nanorods (“AuNRs”) (C12-25-650-TC-50,Nanopartz) were activated to covalently bond amine groups by mixing 40μL of AuNR (4.22×10¹²/mL) with 20 μL of EDC/NHS-sulfo phosphate bufferedsaline (“PBS”) (2 mg/mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1 mg/mL of N-hydroxysulfosuccinimide(Sigma-Aldrich) for 10 minutes at 25° C. These amine-reactive AuNRs werethen PBS-washed and 1 μL of indicated AuNR concentrations were appliedto replicate wells on 192-well amine-functionalized slides (2×10¹²group/mm2, Arrayit), which were sonicated (Q500 Sonicator, Qsonica) for8 minutes at 80% amplitude using a 5 second on/off cycle to acceleratehybridization. Slides were then washed for 10 min at 25° C. with 0.01%Tween-20 in PBS (PBST, pH 7.0), washed with deionized water, and thenair-dried for DFM imagery. Binding affinity was calculated usingnonlinear curve fitting with Origin 2015 software (OriginLabCorporation).

Protein Quantification Assay

Protein A/G-modified 192-well slides (Arrayit) were blocked with 1μL/well Pierce Protein-Free Blocking Buffer (Thermo Scientific) for 1hour at 25° C., then incubated with the indicated amounts ofbiotinylated CD9 antibody (NB110-81616, Novus) for 1 hour at 25° C., andPBS-washed for 10 min at 25° C. before hybridization with AuNR.Neutravidin-functionalized AuNR (Nanopartz C12-25-650-TN-50, 7×10⁻⁹ M)were PBS-diluted (40 μL AuNR to 200 μL PBS) after which 1 μL/well ofAuNR was applied to replicate wells, which were sonicated (Q500Sonicator, Qsonica) for 8 minutes at 80% amplitude using a 5 secondon/off cycle to accelerate hybridization. After hybridization, slideswere washed for 10 min at 25° C. with 0.01% Tween-20 in PBS (PBST, pH7.0), and deionized water, and then air-dried for DFM imagery.

Data Analysis

Limits of detection (LOD) and quantification (LOQ) were defined as 3×and 10× the standard deviation of the assay blank, respectively. Assayprecision was determined with five replicates of three samples analyzedin a single assay (intra-assay) or in three assays analyzed on threedifferent days (inter-assay). Graphs were generated with Origin 2015 andMicrosoft Excel.

Optical Design and Characterization

This hand-held device simply combined (i) a low-cost small triple LEDlight source (˜1k lux), (ii) a dark-field condenser, (iii) a 20× or 10×magnification objective lens, and (iv) 3D printed housings (i.e.,adapter housing and slide housing) to permit the preceding parts tointerface with the mobile phone. To characterize the MDFM apparatus, wecompared this apparatus with a standard DDFM system for imagingnanoparticles. Both systems used the same dark-field condenser, butdiffered in their light sources, objective lenses, cameras, and totalsystem weight and cost.

FIG. 3 provides a comparison of images obtained at high and lowmagnification for biological moieties with and without nanoparticles,respectively, obtained via MDFM, DDFM, and bright field microscopy, with20× and 10× objective lenses employed for MDFM, and with 10× and 4×objective lenses employed for DDFM and bright field microscopy.Comparison of the image quality produced by these systems revealed thatthe MFDM images exhibited weaker and more uneven signals than theequivalent DDFM images, most likely due to their different lightingsystems. The characterized MDFM apparatus employed a relatively weak(1,000-lux) triple-LED light source powered by a 3V lithium battery, asopposed to the DDFM system, which employed a single 100 W (>10,000-lux)halogen lamp. The MDFM light source was chosen for its low power drawand relatively high LED-based light output, but lighting artifactsassociated with the MDFM images are consistent with the three pointangular LED arrangement in the MDFM apparatus. The MDFM condenser,optimized for the DDFM system, may exacerbate these artifacts, as maythe MDFM slide housing, which provides limited precision in aligning thecondenser light path. MDFM image quality also suffers from the lowerresolution of the MDFM CMOS vs. the DDFM CCD sensor (72 vs. 432pixels/inch) at a given objective power. Owing to these differences,different objective lenses were used for the MDFM apparatus (10× and 20×magnification) and DDFM system (4× and 10× magnification) in order tocapture images at similar overall resolution. The low fixed aperture ofthe mobile phone camera (f/2) used by the MDFM apparatus also limitsdepth of focus, but this should not negatively influence surface-basedimagery or biomarker quantification methods.

Functioning as a dark-field, the MDFM apparatus displays thenanoparticle on the surface similar to the DDFM system, as compared tothe bright-field microscope, which was incapable of distinguish thenanoparticle at all. Despite advantages conferred by the more expensiveDDFM system, the MDFM apparatus benefits from the low sensingresolution. The DDFM system with high resolution is more sensitive tosurface scratch and debris, which negatively affect the nanoparticlequantification by imaging. The MDFM apparatus circumvented these noisesby its inherently lower sensing resolution. Moreover, it was determinedthat a primary reason that the MDFM apparatus may be used fornanoparticle quantification is that the sensing resolution does notaffect the quantification result. The magnification may sway thequantification slightly; however, experiments using DDFM under differentresolutions revealed no significant quantification difference.

Another beneficial feature of the MDFM apparatus is its autofocusfunction, which enabled focused 10× magnification DFM images to becaptured over a relatively wide range of working distances (3 to 10mm)—thereby reducing image capture time significantly and allowing theuser to vary the size of the focused images. FIG. 4 shows six MDFMimages captured with a 10× objective lens at different working distancesof 1 mm, 3 mm, 5 mm, 7 mm, 9 mm, and 11 mm, indicating the autofocuslimits of the mobile phone camera used with the MDFM apparatus. FIG. 5is a plot of MDFM magnification (M) with a 10× objective lens versusworking distance (d, mm), with a superimposed linear correlation plotand coefficient of determination (R²) value. A high degree of linearitywas shown. FIG. 6 shows two MDFM images captured with a 20× objectivelens at 2.5 mm and 3.5 mm working distances, with the images beinggrayed to portray the focusing effect.

The MDFM apparatus did not exhibit dynamic working ranges when a 20×magnification objective lens was used, likely due to the more restrictedworking distance available for autofocus. By comparison, the DDFM systemrequired precise manual adjustment to obtain focused images, with a setworking distance and magnification available for each objective lens.The MDFM apparatus magnified samples 370-fold and 110- to 210-fold using20× and 10× objective lenses, while the DDFM system magnified samples375-fold and 150-fold using 10× and 4× objective lenses. After adjustingfor lens and sensor differences, the respective focused high-power andlow-power MDFM images exhibited magnifications corresponding to 98.7%and 58.6% to 112% of their matching DDFM images. It was therefore shownthat the MDFM apparatus is capable of attaining similar magnification asthe DDFM system, while exhibiting more flexibility with respect to theworking distance, and enabling the user to more easily obtain focusedimages. The foregoing features represent significant advantages of aMDFM apparatus in a field setting.

FIG. 10 provides a table comparing features of an exemplary MDFM systemas disclosed herein and a conventional DDFM system. The exemplary MDFMsystem may utilize a three-LED light source providing an aggregateoutput of about 1000 lux, as compared to the DDFM system that utilizes ahalogen lamp providing an output of greater than 10,000 lux. The MDFMsystem may therefore utilize a light source utilizing less than aboutone tenth of the energy and generating less than about one tenth of theheat generated by a light source of a conventional DDFM system. The MDFMsystem may utilize a Motorola XT1064 camera providing 8.0 mexapixelresolution from a CMOS sensor versus an Olympus DP71 camera providing12.5 megapixel resolution from a CCD sensor as utilized by aconventional DDFM system. The MDFM system may entail a total weight 0.38kg and a total cost in a range of $1,360 to $1,560 (depending on theobjective lens used), versus a total eight of 26 kg and a total cost ina range of $50,000. Clearly, the exemplary MDFM system entailsdramatically lower cost and weight than to a conventional DDFM system.

Quantification for Bioassay

To evaluate MDFM performance with common biological assays forquantification, MDFM apparatus and DDFM system results fromnanoparticle-based binding affinity and protein quantitation assays werecompared. FIG. 7A is a schematic view of a binding affinity assay schemeinvolving binding between a carboxyl-acid functionalized gold nanorod(AuNR) and an amine modified slide. The nanoparticle binding assaymeasured the interaction between the carboxylic acid-functionalized goldnanorods (AuNR⁻) and the amine modified slide, with such interactionbeing expressed as a function of AuNR⁻ concentration and the electricfield potential at the liquid-solid interface, which obeys Boltzmannstatistics.

$\begin{matrix}{\left\lbrack {AuNR}^{-} \right\rbrack_{surface} = {\left\lbrack {AuNR}^{-} \right\rbrack_{solution}{\exp\left( \frac{{- e}\;\psi_{D}}{k_{B}T} \right)}}} & (1)\end{matrix}$

where the amount of AuNR available at the slide surface([AuNR⁻]_(surface)) was a function of [AuNR⁻]_(solution), the elementarycharge e (1.60218×10⁻¹⁹ C), the surface potential ψ_(D), the Boltzmannconstant k_(B) (1.38066×10⁻²³ J/K) and temperature T. This equationsimplified to[AuNR⁻]_(surface) =A[AuNR⁻]_(solution)  (2)

when ψ_(D) and T were held constant. Based on the Michaelis-Menten modelat steady-state, the surface binding rate was described as:

$\begin{matrix}{{Response} = \frac{{A\left\lbrack {- {NH}_{3}^{+}} \right\rbrack}_{surface}^{\max} \cdot \left\lbrack {AuNR}^{-} \right\rbrack}{K_{d} + \left\lbrack {AuNR}^{-} \right\rbrack}} & (3)\end{matrix}$

determined by the equilibrium binding constant K_(d), the maximum numberof surface binding sites [NH₃ ⁺]_(surface) ^(max), and the inputnanoparticle concentration constant [AuNR⁻]_(solution), so that K_(d)can be solved for by curve fitting. We applied this information andconcentration-dependent DFM scatter responses from both the MDFMapparatus and DDFM system to calculate the equilibrium binding constant(K_(d)) of this interaction. FIG. 7B provides superimposed plots ofresponse versus nanoparticle concentration (μg/μL) for a bindingaffinity assay according to the scheme of FIG. 7A obtained using a MDFMapparatus as disclosed herein and a conventional DDFM system, withcorresponding nonlinear fitted curves and K_(d) values, wherein datapoints represent the mean±SEM of 5 sample replicates. As shown, the MDFMand DDFM response curves produced in this analysis yielded K_(d) valuesthat did not significantly differ, despite consistently lower MDFMsignal throughout the entire range of the AuNR concentration curve.

Thereafter, MDFM and DDFM performance were analyzed to quantify resultsof a protein binding assay that used protein A/G-modified slides tocapture AuNR-conjugated antibodies. FIG. 8A is a schematic view of aprotein quantification assay scheme involving quantification of abiotinylated antibody aCD9 tagged with an avidin modified goldnanoparticle (AuNR) and bound to a protein A/G modified slide. FIG. 8Bprovides superimposed plots of response versus target concentration(μg/μL) for a protein quantification assay according to the scheme ofFIG. 8A obtained using a MDFM apparatus as disclosed herein and a DDFMsystem, with corresponding linear fits and coefficient of determination(R²) values, and with data points representing the mean±SEM of 5 samplereplicates. Both the MDFM and DDFM responses strongly correlated withthe target protein concentration; however, the MDFM response exhibitedreduced background, less overall variability, and greater linearity thanthe DDFM response. However, the MDFM apparatus also exhibited a smallerdynamic response over the assay concentration range.

Although gold nanorods are disclosed in certain illustrativeembodiments, it is to be appreciated that any suitable nanoparticlesproviding scatter signals may be used in certain embodiments.Non-limiting examples of nanoparticles providing scatter signals includegold or silver nanoparticles. Other types of nanoparticles may be used.

The reduced dynamic range and/or shallower curves of MDFM signals ascompared to DDFM signals in the foregoing assays resulted in higherlimits of detection and quantitation for the MDFM-based assays. FIG. 9is a table identifying sensitivity (including limit of detection (LOD)and limit of quantitation (LOQ) values) for the binding affinity andprotein quantification assays described in connection with FIGS. 7A to8B. The higher limits of detection and quantitation for the MDFM-basedassays corresponded to 7-fold and 4.5-fold sensitivity reductions in thebinding affinity and protein quantification assays. MDFM assays alsorevealed less intra-assay and inter-assay precision than DDFM assays, asreflected by increases in intra-assay (0.9- to 3.2-fold) and inter-assay(1.3- to 3.0-fold) coefficient of variation values. However, the MDFMresults still revealed reasonable coefficients of variation for bothintra-assay (3.1% to 7.8%) and inter-assay (8.1% to 13.7%) replicates.

Lighting induced artifacts observed with the current MFDM prototypeprevent its use for obtaining high-quality DFM imagery, but do notdecrease its utility for nanoparticle-based quantitation assays onceimages are processed to correct for artifacts commonly associated withlow-magnification far-field DFM images (including uneven lighting andother signal artifacts) using a DFM image processing approach.

Differences in MDFM versus DDFM optical performance appears to deriveprimarily from reduced DFM signal quality due to weak and uneven sampleillumination from a multi-LED light source and non-optimized optics inthe exemplary MDFM apparatus disclosed herein. The foregoing issuesshould be easily addressable through selection of a larger,single-source LED and refining the housing (or implementing otherchanges), to improve optical focusing to increase DFM image signalquality. Nevertheless, with easy setup and flexible working distance andmagnification, the MDFM apparatus disclosed herein was effectivelyapplied for the binding affinity and target protein quantificationstudies. MDFM apparatuses as disclosed herein are specificallycontemplated for hand-held or tabletop use in DFM imaging for bioassayquantitation in resource-limited areas in which it would be impracticalor impossible to use conventional DDFM systems.

Those skilled in the art will recognize improvements and modificationsto the preferred embodiments of the present disclosure. All suchimprovements and modifications are considered within the scope of theconcepts disclosed herein and the claims that follow.

What is claimed is:
 1. A dark-field microscope apparatus comprising: anadapter housing configured to receive a portable electroniccommunication device and an objective lens, and to cause the objectivelens to be registered with a camera lens of the portable electroniccommunication device when the portable electronic communication deviceis received by the adapter housing, wherein the adapter housingcomprises a main body and a lens receiver that protrudes from the mainbody, and the lens receiver is configured to receive the objective lens;an electrically operated light source; a dark-field condenser configuredto condense light emissions generated by the electrically operated lightsource; a slide housing configured to receive at least a portion of ananalytical slide and position the analytical slide between thedark-field condenser and the objective lens, wherein the slide housingis configured to receive at least portions of the lens receiver and theobjective lens; a first set screw configured to selectively promoteengagement between the lens receiver and either (i) the slide housing or(ii) the objective lens, to permit adjustment of a first distancebetween the objective lens and the analytical slide; and a second setscrew configured to selectively promote engagement between the slidehousing and the dark-field condenser, to permit adjustment of a seconddistance between the dark-field condenser and the analytical slide. 2.The dark-field microscope apparatus of claim 1, wherein the main bodycomprises a support surface configured to abut a face of the portableelectronic communication device, and the main body comprises at leastone lateral wall configured to abut at least one lateral edge of theportable electronic communication device.
 3. The dark-field microscopeapparatus of claim 1, wherein: the slide housing comprises a bore; thelens receiver comprises a first outer wall configured to fit into afirst portion of the bore; and the dark-field condenser comprises asecond outer wall configured to fit into a second portion of the bore.4. The dark-field microscope apparatus of claim 1, further comprisingthe objective lens.
 5. The dark-field microscope apparatus of claim 4,wherein the objective lens, the slide housing, and the dark-fieldcondenser are configured to form an optical path having a center alignedwith an emissive center of the electrically operated light source. 6.The dark-field microscope apparatus of claim 5, wherein the slidehousing defines at least one slot that is configured to receive the atleast a portion of the analytical slide; and the slide housing isconfigured to permit the analytical slide to move relative to the slidehousing to expose a different portion of the analytical slide to theoptical path with each movement of the analytical slide.
 7. Thedark-field microscope apparatus of claim 4, wherein the objective lensprovides a magnification in a range of 4 times to 100 times.
 8. Thedark-field microscope apparatus of claim 4, wherein the objective lensis configured to provide variable magnification.
 9. The dark-fieldmicroscope apparatus of claim 4, configured to permit the objective lensto be swapped with a different objective lens to provide multipledifferent magnifications.
 10. A biomolecule quantification devicecomprising the dark-field microscope apparatus of claim 4, wherein theanalytical slide is received by the slide housing, and the analyticalslide contains at least one nanoparticle-based biomarker.
 11. Thebiomolecule quantification device of claim 10, wherein the at least onenanoparticle-based biomarker comprises at least one gold or silvernanoparticle.
 12. A method for performing a biological quantitativestudy utilizing the dark-field microscope apparatus according to claim4, the method comprising: inserting at least a portion of the analyticalslide into the slide housing to position the analytical slide betweenthe dark-field condenser and the objective lens, wherein the analyticalslide comprises at least one biomolecule and at least onenanoparticle-based biomarker; transmitting light emissions generated bythe electrically operated light source through the dark-field condenserto impinge condensed light emissions on a target region of the at leasta portion of the analytical slide; and generating a magnified image ofthe target region using the objective lens and the portable electroniccommunication device received by the adapter housing.
 13. A method fordiagnosing a disease using the dark-field microscope apparatus accordingto claim 4, the method comprising: inserting at least a portion of theanalytical slide into the slide housing to expose an area of interest ofthe analytical slide to an optical path between the dark-field condenserand the objective lens, wherein the area of interest of the analyticalslide contains target biomolecules and labels embodying conjugatednanoparticles comprising binding counterparts for the targetbiomolecules; transmitting light emissions generated by the electricallyoperated light source through the dark-field condenser to impingecondensed light on the area of interest of the analytical slide; andgenerating a magnified image of the area of interest of the analyticalslide using the objective lens and the portable electronic communicationdevice received by the adapter housing; and analyzing the magnifiedimage.
 14. The dark-field microscope apparatus of claim 1, furthercomprising a base element configured to support the electricallyoperated light source and configured to receive an end portion of theslide housing.
 15. The dark-field microscope apparatus of claim 1,wherein the electrically operated light source comprises a solid statelight source.
 16. The dark-field microscope apparatus of claim 15,wherein the solid state light source comprises at least one lightemitting diode configured to generate a peak wavelength in the visiblerange.
 17. The dark-field microscope apparatus of claim 1, wherein theportable electronic communication device comprises a mobile phone. 18.The dark-field microscope apparatus of claim 1, wherein the adapterhousing comprises at least one polymeric material and the slide housingcomprises at least one polymeric material.
 19. The dark-field microscopeapparatus of claim 1, wherein the adapter housing and the slide housingare fabricated by three-dimensional printing.
 20. The dark-fieldmicroscope apparatus of claim 1, wherein the adapter housing and theslide housing are fabricated by molding.
 21. The dark-field microscopeapparatus of claim 1, wherein the adapter housing and the slide housingare fabricated by a subtractive material removal process.